COP9 Signalosome is a multi-functional protein complex conserved from yeast to humans. It plays an essential role in diverse cellular functions by regulating a large family of cellular machines named cullin- based ubiquitin ligases. The cullin-based ubiquitin ligase machinery promotes the attachment of a polyubiquitin chain to many regulatory and signaling protein substrates to target them for proteasome- mediated degradation. By controlling the protein level of these substrates, cullin-based ubiquitin ligases can specifically regulate various cellular events in cell cycle progression, intracellular signaling, DNA replication and repair, and gene transcription. De-regulations of cullin-based ubiquitin ligases have been implicated in a number of human diseases such as tumorigenesis, HIV infection, and neurological disorders. Recent studies have suggested that COP9 Signalosome might coordinate the assembly of the cullin-based ubiquitin ligase machinery through modulating the post-translational modification of the cullin scaffold proteins by a ubiquitin-like molecule, NeddS. A complete mechanistic understanding of the COP9 Signalosome's functions, however, requires detailed structural analyses and structure-based functional characterization of the complex. This proposal aims to develop a novel method for producing large quantities of recombinant COP9 Signalosome, which is required for crystallization of the complex. Our preliminary studies have shown that a number of the COP9 Signalosome subunits, when over-expressed in bacteria, are either insoluble or unstable. To tackle these problems, we plan to simultaneously co-express all eight subunits of the COP9 Signalosome complex in both bacteria and insect cells. This approach has already allowed us to produce a soluble and stable COP9 sub-complex. In addition, we will carry out biochemical analyses of the recombinant COP9 Signalosome complex to test its cullin-binding and cullin deneddylation activities. The long-term goal of this research is to determine the crystal structure of the full COP9 Signalosome complex in order to reveal the structural basis of its functions, in particular, its regulation of the cullin-based ubiquitin ligase machinery in the context of various cellular pathways. [unreadable] [unreadable] [unreadable]